Antigen presentation by MHC class I molecules to CD8 T cells is a major pathway by which the acquired immune system detects and eliminates virus infected cells. All nucleated cells express MHC class I molecules and are thus potentially capable of direct antigen presentation to CD8 T cells upon infection. However, several, but not all, recent studies suggest that predominantly DCs (or a specific subset of DCs) are uniquely required for in vivo priming of CD8 T cells to virus. Because pathogens may not directly infect these requisite DCs, cross presentation pathways have been proposed;in essence, the infected cell may not be the primary antigen presenting cell. Additionally, for many arthropod-transmitted viruses, virus-specific antigens may require transfer from migratory DCs in the skin to lymph node resident DCs to efficiently prime CD8 T cells. However, the mechanism by which pathogen-specific antigens are shuttled from the infected cells to DCs or between DC subsets is unknown. Not surprisingly, these same issues of direct presentation vs. cross-presentation also apply to CD8 T cell responses to tumors or following DNA vaccination. As a novel strategy to elicit pathogen immunity, we have engineered preprocessed and preloaded MHC class I molecules as single chains of peptide, beta-2 microglobulin and class I heavy chain. We have termed these complexes single chain trimers or SCTs. SCTs are very stably expressed at the cell surface and we and others have demonstrated that SCTs elicit a robust CD8 T cell response. In this grant we will test whether SCTs confer protective immunity against viruses and bacteria, and probe the cellular and molecular basis of vivo priming of CD8 T cells following SCT vaccination. Our hypothesis is that SCT vaccine efficacy results from crosspresentation by CD81 DCs using a novel mechanism involving intercellular membrane exchange. PUBLIC HEALTH RELEVANCE: Protective immunity to several pathogens requires that MHCI molecules bind antigenic peptides for presentation to CD8 T cells. However, to bind MHCI molecules, pathogen-derived peptides must compete with an extensive pool of endogenous peptides of the host. We have engineered MHCI molecules so that they can be pre-loaded with pathogen-derived peptides. In this grant, we will test the efficacy and mechanism of using these pre-loaded MHCI as vaccines.